iCLIP - transkriptom hela Kartläggning av protein-RNA interaktioner med individuella Nukleotid Upplösning

Biology
 

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Summary

Den rumsliga arrangemang av RNA-bindande proteiner på en utskrift är en viktig faktor för post-transkriptionell reglering. Därför utvecklade vi individuella nukleotida crosslinking upplösning UV och immunoprecipitation (iCLIP) som ger en exakt genomet hela kartläggning av bindningar på ett RNA-bindande protein.

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Konig, J., Zarnack, K., Rot, G., Curk, T., Kayikci, M., Zupan, B., Turner, D. J., Luscombe, N. M., Ule, J. iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution. J. Vis. Exp. (50), e2638, doi:10.3791/2638 (2011).

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Abstract

Den unika sammansättning och rumsliga arrangemang av RNA-bindande proteiner (RBPs) på en avskrift styra olika aspekter av post-transkriptionell reglering 1. Därför är ett viktigt steg mot att förstå avskrift reglering på molekylär nivå för att få positionsinformation om bindningar på RBPs 2.

Protein-RNA interaktioner kan studeras med biokemiska metoder, men dessa metoder inte adressen RNA bindande i dess ursprungliga cellulära sammanhang. Inledande försök att studera protein-RNA komplex i sin cellulära miljö anställd affinitet rening eller immunoprecipitation kombinerat med differential display eller microarray analys (RIP-CHIP) 3-5. Dessa metoder var benägna att identifiera indirekta eller icke-fysiologiska samspel 6. För att öka specificiteten och positionella upplösning, som en strategi för att så CLIP (UV tvärbindning och immunoprecipitation) infördes 7,8. CLIP kombinerar UV tvärbindning av proteiner och RNA-molekyler med rigorösa rening system inklusive denaturering polyakrylamidgelelektrofores. I kombination med hög genomströmning sekvensering teknik, har CLIP visat sig vara ett kraftfullt verktyg för att studera protein-RNA interaktioner på en genomet-omfattande (kallas HITS-klipp eller CLIP-punkter) 9,10. Nyligen var PAR-CLIP infördes som använder fotoreaktiva ribonucleoside analoger för förnätning 11,12.

Trots den höga specificitet av erhållna data, CLIP experiment leder ofta till cDNA bibliotek av begränsad sekvens komplexitet. Detta beror delvis på den begränsade mängden co-renade RNA och två ineffektiva RNA reaktioner ligation krävs för biblioteket beredning. Dessutom visade primer förlängning analyser som många cDNAs trunkera förtid på tvärbunden nukleotid 13. Sådana trunkerad cDNAs försvinner under standarden CLIP biblioteket förberedelse protokollet. Vi utvecklade nyligen iCLIP (individ-nucleotide upplösning CLIP), som fångar den trunkerade cDNAs genom att ersätta en av de ineffektiva intermolekylära RNA ligation steg med en mer effektiv intramolekylära cDNA circularization (Figur 1) 14. Viktigt sekvensering den trunkerade cDNAs ger insikter i position över länken plats hos nukleotid upplösning. Vi tillämpats med framgång iCLIP att studera hnRNP C partikel organisation på ett genomet-omfattande och bedöma dess roll i skarvning Regel 14.

Protocol

1. UV bryggbindningen av vävnadsodling celler

  1. Ta bort materialet och lägga till 6 ml iskall PBS till celler odlas i en 10 cm tallrik (räcker till tre experiment).
  2. Ta bort locket och placera på is. Bestråla en gång med 150 mJ / cm 2 vid 254 nm.
  3. Harvest cellerna genom att skrapa med en cell lyftare.
  4. Överför 2 ml cellsuspension vardera till tre mikrorör. Snurra på högsta hastighet i 10 sek vid 4 ° C till pellets celler, ta sedan bort supernatanten.
  5. Snap-frysa cellen pellets på torris och förvaras vid -80 ° C fram till användning.

2. Bead förberedelse

  1. Tillsätt 100 ìl av protein A Dynabeads (Dynal, 100,02) per försök till en ny mikrorör (Använd protein G Dynabeads för en mus eller antikroppar get).
  2. Tvätta pärlor 2x med lyseringsbuffert (50 mM Tris-HCl, pH 7,4, 100 mM NaCl, 1% NP-40, 0,1% SDS, 0,5% natriumdeoxikolat, 1 / 100 proteashämmare cocktail III Calbiochem).
  3. Återsuspendera pärlor i 100 l lyseringsbuffert med 2-10 mikrogram antikropp.
  4. Rotera rören i rumstemperatur i 30-60 min.
  5. Tvätta 3x med 900 l lyseringsbuffert och lämna in den sista tvättningen tills de ska gå vidare till steg 4,1.

3. Cellslys och partiell RNA matsmältning

  1. Resuspendera cellpelleten i 1 ml lyseringsbuffert och överför till 1,5 ml mikrorör.
  2. Bered en 1 / 500 utspädning av RNas I (Ambion, AM2295). Tillsätt 10 l RNas jag utspädning samt 2 l Turbo DNas till cellen lysat (1 / 500 RNas jag spädningar [låg RNas] används för biblioteket beredning, 1 / 50 utspädning [hög RNas] är nödvändiga för att kontrollera för antikroppar specificitet) .
  3. Inkubera prov för exakt 3 min vid 37 ° C, skaka vid 1100 rpm. Överför omedelbart till is.
  4. Spin vid 4 ° C och 22.000 gi 20 minuter för att rensa lysat. Försiktigt samla supernatanten (lämna ca 50 l lysat med pellets).

4. Immunoprecipitation

  1. Ta bort tvättbuffert från pärlor (från steg 2,5), lägg sedan till cellen lysat (från steg 3,4).
  2. Rotera prover för 2 h vid 4 ° C.
  3. Kasta bort vätskefasen och tvätta pärlor 2x med 900 l hög saltbuffert (50 mM Tris-HCl, pH 7,4, 1 M NaCl, 1 mM EDTA; 1% NP-40, 0,1% SDS, 0,5% natriumdeoxikolat).
  4. Tvätta 2x med 900 l tvättbuffert (20 mM Tris-HCl, pH 7,4, 10 mM MgCl 2, 0,2% Tween-20).

5. Defosforylationen av RNA 3'ends

  1. Kassera supernatanten och suspendera pärlor i 20 l PNK blandning (15 l vatten, 4 l 5x PNK pH 6,5 buffert [350mMTris-HCl, pH 6,5; 50mMMgCl 2 25mMdithiothreitol], 0,5 l PNK enzym, 0,5 l RNasin [Promega]).
  2. Inkubera 20 minuter vid 37 ° C.
  3. Tillsätt 500 l tvättbuffert och tvätta 1x med hög salt-buffert.
  4. Tvätta 2x med tvättbuffert.

6. Länkaren ligatur till RNA 3 "slutar

  1. Ta försiktigt bort supernatanten och återsuspendera pärlor i 20 l ligation mix (9 l vatten, 4 l 4x ligation buffert [200 mMTris-HCl, 40m MM GCL 2, 40 mM ditiotreitol], 1 l-RNA ligas [NEB], 0,5 l RNasin [Promega], 1,5 l före adenylated länkare L3 [20 mikroM], 4 l PEG400 [81.170, Sigma]).
  2. Inkubera över natten vid 16 ° C.
  3. Tillsätt 500 l tvättbuffert och tvätta sedan 2x med 1 ml hög salt-buffert.
  4. Tvätta 2x med 1 ml tvättbuffert och lämna i 1 ml av den andra tvätten.

7. RNA 5 'slut märkning

  1. Avlägsna supernatanten och suspendera pärlor i 8 l varmt PNK mix (0,4 l PNK [NEB], 0,8 l 32 P-γ-ATP, 0,8 l 10x PNK buffert [NEB], 6 l vatten).
  2. Inkubera 5 minuter vid 37 ° C.
  3. Ta ut den varma PNK mixen och resuspendera pärlor i 20 l 1x Nupage buffert (Invitrogen).
  4. Inkubera på en thermomixer vid 70 ° C i 10 min.
  5. Placera omedelbart på en magnet för att fälla den tomma pärlor och ladda supernatanten på gelen (se steg 8).

8. SDS-PAGE och membran överföra

  1. Ladda prover på en 4-12% NuPAGE Bis-Tris gel (Invitrogen) enligt tillverkarens anvisningar. Använd 0,5 l 1x MOPS löpande buffert (Invitrogen). Även belastning 5 ìl av en pre-färgad protein storlek markör (till exempel PAGE härskare plus, Fermentas, SM1811).
  2. Kör gelen i 50 min vid 180 V.
  3. Ta bort gelen fram och kasta som fast avfall (innehåller fri radioaktiva ATP).
  4. Överför protein-RNA komplex från gelen till ett nitrocellulosa membran med Novex våta överföringen apparater enligt tillverkarens anvisningar (Invitrogen, överför 1 h vid 30 V).
  5. Efter överföringen, skölj membranet i PBS-buffert, sedan linda in det i Saran wrap och utsätt det för en Fuji film på -80 ° C (placera en fluorescerande klistermärke intill membranet att senare anpassa than film och membranet, utföra exponering i 30 min, 1 timme och över natten).

9. RNA-isolering

  1. Isolera protein-RNA komplex från låg-RNas experimentera med autoradiograph från steg 8,5 som en mask. Klipp denna bit av membranet i flera små skivor och placera dem i en 1,5 ml mikrorör.
  2. Tillsätt 200 l PK buffert (100 mM Tris-HCl pH 7,4, 50 mM NaCl, 10 mM EDTA) och 10 l proteinas K (Roche 03115828001) till membranet bitar. Inkubera skaka vid 1100 rpm i 20 minuter vid 37 ° C.
  3. Tillsätt 200 ìl PKurea buffert (100 mM Tris-HCl pH 7,4, 50 mM NaCl, 10 mM EDTA, 7 M urea) och inkubera i 20 minuter vid 37 ° C.
  4. Samla lösning och tillsätt det tillsammans med 400 l av RNA-fenol / kloroform (Ambion, 9722) till en 2 ml Phase Lock Gel Tunga rör (713-2536, VWR).
  5. Inkubera 5 minuter vid 30 ° C, skaka vid 1100 rpm. Separera faserna genom att snurra i 5 min vid 13.000 rpm vid rumstemperatur.
  6. Överför vattenskiktet i en ny tub (var försiktig att inte röra gelen med pipett). Tillsätt 0,5 l glycoblue (Ambion, 9510) och 40 l 3 M natriumacetat pH 5,5 och blanda. Tillsätt sedan 1 ml 100% etanol, blanda igen och fällningen över natten vid -20 ° C.

10. Omvänd transkription

  1. Spin i 20 min vid 15.000 rpm och 4 ° C. Avlägsna supernatanten och tvätta pelleten med 0,5 ml 80% etanol.
  2. Återsuspendera pelleten i 7,25 l RNA / primermix (6,25 l vatten, 0,5 l Rclip primer [0.5pmol/μl], 0,5 l dNTP mix [10 mM]). För varje experiment eller kopiera, använda en annan Rclip primer innehåller enskilda streckkod sekvenser (se 14).
  3. Inkubera 5 minuter vid 70 ° C innan kylning till 25 ° C.
  4. Tillsätt 2,75 l RT blandning (2 l 5x RT-buffert, 0,5 l 0,1 miljoner DTT, 0,25 l Upphöjd III omvänt transkriptas [Invitrogen]).
  5. Inkubera 5 minuter vid 25 ° C, 20 minuter vid 42 ° C, 40 min vid 50 ° C och 5 min vid 80 ° C innan kylning till 4 ° C.
  6. Tillsätt 90 l TE buffert, 0,5 l glycoblue och 10 l natriumacetat pH 5,5 och blanda. Tillsätt sedan 250 l 100% etanol, blanda igen och fällningen över natten vid -20 ° C.

11. Gel rening av cDNA

  1. Spin ner och tvätta proverna (se 10.1), sedan resuspendera pellets i 6 l vatten.
  2. Tillsätt 6 l 2x TBE-urea buffert (Invitrogen). Värm prover till 80 ° C i 3 min direkt före lastning.
  3. Ladda prover på en prefabricerad 6% TBE-urea gel (Invitrogen) och kör i 40 min vid 180 V som enligt tillverkaren. Också ladda en låg molekylvikt markör för efterföljande skärning (se nedan).
  4. Skär tre band på 120-200 NT (hög), 85-120 NT (medium) och 70-85 NT (låg). Använd theupper färgämnet och märken på plasten gelen stöd för att vägleda excision (se figur 3). Observera att Rclip primer och L3 sekvensen tillsammans står för 52 nt av klippet sekvensen.
  5. Tillsätt 400 l TE och krossa gelen skär i små bitar med hjälp av en 1 ml spruta kolven. Inkubera skaka vid 1100 rpm under 2 h vid 37 ° C.
  6. Placera två 1 cm glas förfilter (Whatman, 1.823.010) till en Costar Spinx kolumn (Corning Incorporated, 8161). Överför vätskan del av provet till kolonnen. Spin i 1 min vid 13 tusen varv i ett 1,5 ml rör.
  7. Tillsätt 0,5 l glycoblue och 40 l natriumacetat pH 5,5, blanda provet. Tillsätt 1 ml 100% etanol, blanda igen och fällningen över natten vid -20 ° C.

12. Ligatur grundfärg till 5'end av cDNA

  1. Spin ner och tvätta proverna (se 10.1), sedan resuspendera pellets i 8 l ligation mix (6,5 l vatten, 0,8 l 10x CircLigase Buffer II, 0,4 l 50 mM MnCl 2, 0,3 l, Circligase II [Epicentrum]) och inkubera för 1 h vid 60 ° C.
  2. Tillsätt 30 l oligo glödgning blandning (26 l vatten, 3 l FastDigest buffert [Fermentas], 1 l cut_oligo [10 mikroM]). Inkubera i 1 min vid 95 ° C. Sedan minska temperaturen var 20 sek med 1 ° C tills 25 ° C uppnås.
  3. Tillsätt 2 l BamHI (Fast Fermentas) och inkubera i 30 minuter vid 37 ° C.
  4. Tillsätt 50 l TE och 0,5 l glycoblue och blanda. Tillsätt 10 l pH natriumacetat 5,5 och blanda, lägg sedan till 250 l 100% etanol. Blanda igen och fällningen över natten vid -20 ° C.

13. PCR-amplifiering

  1. Spin ner och tvätta proverna (se 10.1), sedan suspendera pelleten i 19 l vatten.
  2. Bered PCR-mix (19 l cDNA, 1 l primermix P5/P3 Solexa, 10 mikroM vardera, 20 l Accuprime Supermix ett enzym [Invitrogen]).
  3. Kör följande PCR-program: 94 ° C under 2 min, [94 ° C i 15 sekunder, 65 ° C under 30 sek, 68 ° C i 30 sek] 25-35 cykler, 68 ° C i 3 min, 4 ° C för alltid.
  4. Blanda 8 l PCR-produkten med 2 ìl 5x TBE-buffert och last på en prefabricerad 6% TBE gel (InvitRogen). Fläck gelen med Sybrgreen I (Invitrogen) och analysera med en gel värmekamera.
  5. Streckkoden i Rclip primers låt multiplex olika prover innan du skickar in för hög genomströmning sekvensering. Skicka in 15 ìl av biblioteket för sekvensering och spara resten.

14. Länkare och primer sekvenser

Pre-adenylated 3 "länkare DNA:

[Vi beställer DNA adaptern från IDT och sedan göra portioner av 20 mikroM.]

DNA

15. Representativa resultat:

Innan sekvensering av iCLIP biblioteket kan framgång försöket övervakas på två steg: autoradiograph av protein-RNA komplex efter membran överföring (steg 8,5) och den bild av gelen av PCR-produkter (steg 13,4). I autoradiograph av låg-RNase stickprov bör diffusa radioaktivitet ses över molekylvikt av proteinet (Figur 2, exempel 4). För hög RNase prover, detta är radioaktivitet fokuserat närmare molekylvikt av proteinet (Figur 2, exempel 3). När ingen antikroppar används i immunoprecipitation bör ingen signal detekteras (Figur 2, exempel 1 och 2). Ytterligare viktiga kontroller för specificitet immunoprecipitation antingen utelämna UV-strålning eller celler använder som inte uttrycker proteinet av intresse 14.

Gelen bilden av PCR-produkterna (steg 13,4) ska visa en storleksordning som motsvarar cDNA fraktion (hög, medel eller låg) renas i steg 11,4 (Figur 4, banor 4-6). Observera att PCR-primers P3Solexa och P5Solexa införa ytterligare 76 NT till storleken på cDNA. Om inga antikroppar används under immunoprecipitation bör inga motsvarande PCR-produkterna kan detekteras (Figur 4, banor 1-3). Primer dimer produkt kan visas på ca 140 NT.

För representativa resultat av hög genomströmning sekvensering och efterföljande bioinformatiska analyser se 14.

Figur 1
Figur 1. Schematisk representation av iCLIP protokollet. Protein-RNA komplex är kovalent tvärbunden in vivo med hjälp av UV-strålning (steg 1). Proteinet av intresse renas tillsammans med bundna RNA (steg 2-5). För att möjliggöra sekvensspecifika priming av omvänd transkription, är ett RNA-adapter knyts ihop till 3 'änden av RNA, medan 5 "slutet är radioaktivt märkt (steg 6 och 7). Tvärbunden protein-RNA komplex är renade från gratis RNA med hjälp av SDS-PAGE och membran överföring (steg 8). RNA återvinns från membranet genom uppslutning av proteinet med proteinas K lämnar en polypeptid kvar på korset-link nukleotid (steg 9). Omvänd transkription (RT) trunkerar på återstående polypeptid och introducerar två cleavable adaptern regioner och sekvenser streckkod (steg 10). Val av storlek tar bort fri RT primer innan circularization. Följande linjärisering genererar lämpliga mallar för PCR-amplifiering (steg 11-15). Slutligen genererar hög genomströmning sekvensering läser där streckkoden sekvenser omedelbart följs av den sista nukleotid av cDNA (steg 16). Eftersom detta nukleotid lokaliserar ett läge uppströms tvärbunden nukleotid kan bindningsstället härledas med hög upplösning.

Figur 2
Figur 2. Autoradiograph av tvärbunden hnRNP C-RNA komplex med denaturering gelelektrofores och membran överföring. hnRNP C-RNA komplex var immun-renat från cell extrakt med hjälp av en antikropp mot hnRNP C (α hnRNP C, prov 3 och 4). RNA var delvis smälta med låg (+) eller hög (+ +) koncentration av RNas. Komplex förskjutning uppåt från storleken på protein (40 kDa) kan observeras (prov 4). Övergången är mindre uttalad vid höga koncentrationer av RNas användes (prov 3). Det radioaktiva signalen försvinner när ingen antikroppar användes i immunoprecipitation (prov 1 och 2).

Figur 3
Figur 3. Schematisk 6% TBE-urea gel (Invitrogen) för att styra excision av iCLIP cDNA produkter. Gelen körs för 40 min vid 180 V vilket leder till en reproducerbar migration mönster av cDNAs och färgämnen (ljust och mörkt blått) i gelen. Använd ett rakblad för att skära (röda linjen) till hög (H), medel (M) och låg (L) cDNA bråk. Börja med att skära i mitten av ljusblå färg och ovanför märket på plast gelen kassetten. Dela medium och låg fraktioner och trimma hög andel ca 1 cm ovanför ljusblå färg. Använd vertikalt snitt styrs av fickorna och färgen att skilja de olika köer (i detta exempel 1-4). Markören Lane (m) kan färgas och avbildas för att styrastorlekar efter kapning. Fragment storlekar anges till höger.

Figur 4
Figur 4. Analys av PCR-förstärkta iCLIP cDNA bibliotek med hjälp av gelelektrofores. RNA återhämtat sig från membranet (Figur 1) var omvänd transkriberat och storlek-renas med hjälp av denatureringen gelelektrofores (Figur 2). Tre stora delar av cDNA (hög [H]: 120-200 nt, medium [M]: 85-120 nt och låga [L]: 70-85 NT) omhändertogs, circularized, re-linjär och PCR-förstärkning. PCR-produkter av olika storleksfördelning kan observeras som ett resultat av de olika storlekarna av den ingående fraktioner. Eftersom PCR primern introducerar 76 nt till cDNA, bör storlek varierar mellan 196-276 nt för hög, 161-196 nt för medelstora och 146-161 NT för liten storlek fraktioner. PCR-produkterna är frånvarande när ingen antikroppar har använts för immunoprecipitation (körfält 1-3).

Discussion

Eftersom iCLIP protokollet innehåller ett varierat utbud av enzymatiska reaktioner och steg rening, är det inte alltid lätt att identifiera ett problem när ett experiment misslyckas. För att kontrollera för specificitet identifierade RNA cross-link webbplatser bör en eller flera negativa kontroller hållas under hela försöket och efterföljande beräkningar analyser. Dessa kontroller kan vara no-antikroppen prov, den icke-tvärbundet celler, eller immunoprecipitation från knockout-celler eller vävnader. Helst bör dessa kontrollmekanismer experiment rena inga protein-RNA komplex, och därför bör ge någon signal på SDS-PAGE gel, och ingen mätbar produkter efter PCR-amplifiering. Hög genomströmning sekvensering av dessa kontroll biblioteken bör återgå mycket få unika sekvenser. ÖVERVÄLDIGANDE celler rekommenderas inte som en sekvensering kontroll, eftersom den resulterande sekvenser fortfarande motsvarar över Länk sajter av samma protein, som är renat från ÖVERVÄLDIGANDE celler i mindre kvantiteter.

Försiktighetsåtgärder bör också vidtas för att undvika kontaminering med PCR-produkter från tidigare experiment. Det bästa sättet att minimera detta problem är att rumsligt åtskilda pre-och post-PCR steg. Helst bör en analys av PCR-produkter och alla efterföljande steg ska utföras i ett separat rum. Vidare bör varje medlem i laboratoriet använder sin egen uppsättning av buffertar och andra reagenser. På detta sätt kan föroreningskällor vara lättare identifieras.

Disclosures

Inga intressekonflikter deklareras.

Acknowledgements

Författarna tackar alla medlemmar i Ule, Luscombe och Zupan laboratorier för diskussion och experimentell hjälp. Vi tackar James Hadfield och Nik Matthews för hög genomströmning sekvensering. Vi vill påpeka att iCLIP metod som beskrivs här aktierna flera steg med den ursprungliga CLIP-protokollet, som utvecklats av Kirk Jensen och HJ i laboratorium av Robert Darnell. Detta arbete stöddes av Europeiska forskningsrådet bevilja 206.726-klippet till HJ och en Långsiktig Human Frontiers Science Program gemenskap till JK

Materials

Name Company Catalog Number Comments
For gel electrophoresis and membrane transfer we recommend t he use of XCell SureLock® Mini-Cell and XCell IIâ Blot Module Kit CE Mark (Invitrogen, EI0002), which is compatible with the use of the different precast minigels that are specified throughout the protocol. The brand and order number of all materials used is mentioned during the protocol. The list of enzymes used in the protocol is shown in the table below.
Protein A Dynabeads Invitrogen 10001D use protein G for mouse or goat antibody
RNase I Ambion AM2295 activity can change from batch to batch
T4 RNA ligase I New England Biolabs M0204S
PNK New England Biolabs M0201S
proteinase K Roche Group 03115828001
Superscript III reverse transcriptase Invitrogen 18080044
Circligase II Epicentre Biotechnologies CL9021K
FastDigest® BamHI Fermentas FD0054
AccuPrime™ SuperMix I Invitrogen 12342010 this PCR mix gives the best results in our hands

DOWNLOAD MATERIALS LIST

References

  1. Keene, J. D. RNA regulons: coordination of post-transcriptional events. Nat Rev Genet. 8, 533-543 (2007).
  2. Wang, Z., Burge, C. B. Splicing regulation: from a parts list of regulatory elements to an integrated splicing code. RNA. 14, 802-813 (2008).
  3. Trifillis, P., Day, N., Kiledjian, M. Finding the right RNA: identification of cellular mRNA substrates for RNA-binding proteins. RNA. 5, 1071-1082 (1999).
  4. Brooks, S. A., Rigby, W. F. Characterization of the mRNA ligands bound by the RNA binding protein hnRNP A2 utilizing a novel in vivo technique. Nucleic Acids Res. 28, E49-E49 (2000).
  5. Tenenbaum, S. A., Carson, C. C., Lager, P. J., Keene, J. D. Identifying mRNA subsets in messenger ribonucleoprotein complexes by using cDNA arrays. Proc Natl Acad Sci. 97, 14085-14090 (2000).
  6. Mili, S., Steitz, J. A. Evidence for reassociation of RNA-binding proteins after cell lysis: implications for the interpretation of immunoprecipitation analyses. RNA. 10, 1692-1694 (2004).
  7. Ule, J. CLIP identifies Nova-regulated RNA networks in the brain. Science. 302, 1212-1215 (2003).
  8. Ule, J., Jensen, K., Mele, A., Darnell, R. B. CLIP: A method for identifying protein-RNA interaction sites in living cells. Methods. 37, 376-386 (2005).
  9. Licatalosi, D. D. HITS-CLIP yields genome-wide insights into brain alternative RNA processing. Nature. 456, 464-469 (2008).
  10. Yeo, G. W. An RNA code for the FOX2 splicing regulator revealed by mapping RNA-protein interactions in stem cells. Nat Struct Mol Biol. 16, 130-137 (2009).
  11. Urlaub, H., Hartmuth, K., Lührmann, R. A two-tracked approach to analyze RNA-protein crosslinking sites in native, nonlabeled small nuclear ribonucleoprotein particles. Methods. 26, 170-181 (2002).
  12. König, J. iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution. Nat Struct Mol Biol. 17, 909-915 (2010).

Erratum

Formal Correction: Erratum: iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution
Posted by JoVE Editors on 07/14/2011. Citeable Link.

A correction was made to iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution. There was an error in part 2 of step 3. One of the characters had the incorrect symbol and was corrected to:

"...as well as 2 μl Turbo DNase..."

instead of:

"...as well as 2 ml Turbo DNase..."

Comments

71 Comments

  1. Hi,

    First I would like to say this latest method is really neat. I also like Julian's comment at the end of the video when he said with a big smirk, "You have to perform each of the 64 steps with 100% accuracy". :D That is epic.

    On a more serious note, I am just wondering if anyone can suggest what sort of primer I should use if I want to start by cloning my insert into TOPO vector instead of doing nextGen sequencing. Any help is appreciated.

    Paul

    Reply
    Posted by: Anonymous
    June 9, 2011 - 3:47 AM
  2. Hi Paul, thanks for your fun comment! TOPO cloning dŒsn²17;t require any specific primer, so you could use the one described in the protocol. Unless you wish to do something specific, such as concatemerization of sequences before inserting them into vector. Feel free to post more questions! Jernej

    Reply
    Posted by: Anonymous
    June 11, 2011 - 4:19 PM
  3. For more iCLIP questions and answers, use the following Googledoc: http://goo.gl/4tSci.

    Reply
    Posted by: Anonymous
    June 13, 2011 - 11:28 AM
  4. Hi Jernej,
    Is it possible to use a 3' linker with a phosphorylated 5' end instead of a pre-adenylated 5' end and adding some ATP during the 3' linker ligation step? Thanks. Paul

    Reply
    Posted by: Anonymous
    June 13, 2011 - 10:13 PM
  5. Yes, just follow the protocol as described in Konig et al, NSMB ²010 (PMID ²0601959). More on Googledoc.

    Reply
    Posted by: Anonymous
    June 14, 2011 - 3:48 AM
  6. Hi Jernej,

    Sorry to keep bombarding you with questions. In the supplementary section of your NSMB ²010 paper, shrimp alkaline phosphatase was used to desphosphorylate 3' ends. My understanding is that SAP can only desphosphorylate 5' ends. I am wondering if you had dephosphorylated 3' ends step using PNK before using SAP to dephosphorylate 5' ends.

    Quote from Konig et al, NSMB ²010: "For dephosphorylation of 3²4²; ends, Dynabeads were resuspended in ² µl 10&#²15; Shrimp alkaline phosphatase buffer (Promega), 17.5 µl H²O and 0.1 µl Shrimp alkaline
    phosphatase (Promega) and incubated at 37°C for 10 min with intermittent shaking (10 sec at 700 rpm followed by ²0 sec pause)."

    Thank you again for your help.

    Reply
    Posted by: Anonymous
    October 5, 2011 - 4:04 AM
  7. We did use SAP in the NSMB protocol - it dŒsn't work as well as PNK on the 3' ends. We couldn't use PNK at the time, because PNK carryover into ligation reaction would create problems in the presence of ATP. In jove protocol, ligation reaction lacks ATP, therefore we can use PNK to dephosphorylate the 3' ends.

    Reply
    Posted by: Anonymous
    October 5, 2011 - 6:30 PM
  8. Hi Jernej,

    On 3.² it says add ²ml Turbo DNAse into the 1.5 ml tube. I am wondering if that amount is correct.

    Reply
    Posted by: Anonymous
    July 9, 2011 - 11:45 PM
  9. Hello Paul,
    you are right, it should be two micro liters. Sorry for that, I will try to have it changed,
    Julian

    Reply
    Posted by: Anonymous
    July 10, 2011 - 6:56 AM
  10. Hi Jernej, great protocol! Just a precision, the L3 oligo is a pre-adenylated DNA or RNA oligo? Not clear as the original Clip and iClip uses RNA...

    Thanks a bunch,

    Marco

    Reply
    Posted by: Anonymous
    September 12, 2011 - 3:58 PM
  11. Hi Marco. It's a DNA oligo. Best, Jernej

    Reply
    Posted by: Anonymous
    September 12, 2011 - 4:02 PM
  12. Hi Jernej,

    I am wondering if the 3²P-ATP batch that you normally use in your lab for step 6.1 always has close to a 100% reported radioactivity. What is the lowest percentage of remaining 3²P that you can usually still get away with? I can still get some decent signal when using 3²P-ATP that has ~50-60% remaining radioactivity but my bands on the films are not as intense as the one that I see in your publications. I am trying to work out the best schedule for ordering some 3²P-ATP and starting my experiments. Thanks again.

    Paul

    Reply
    Posted by: Anonymous
    September 14, 2011 - 11:32 PM
  13. We don't use ATP if it's more than two weeks old, thus we have >50% radioactivity. But signal intensity also depends on the efficiency of crosslinking and IP,and amount of protein expression in the cells.

    Reply
    Posted by: Anonymous
    September 15, 2011 - 4:23 AM
  14. Hi Jernej,
    Thank you for the protocol. What results if I reduce the cell samples to 100-1000 (not 10*6-7 cells) ? Thanks for your reply.

    Reply
    Posted by: Anonymous
    November 15, 2011 - 12:24 AM
  15. That would be challenging. If you have an abundant protein that cross-links well to RNA, then it might be possible. So try running the radioactive protein-RNA complex on the gel - if you good signal after overnight exposure, then it's doable.

    Reply
    Posted by: Anonymous
    November 15, 2011 - 4:52 AM
  16. Hi, Jernej !
    Thank you for the reply. I have another questions: How stable if the RNA-RNA and RNA-Protein photocrosslinking? How to degrade these proteins or remove the photocrosslinking? Thank you a lots.

    Reply
    Posted by: Anonymous
    November 15, 2011 - 6:36 AM
  17. Hi Jernej,

    I again have some more questions. Do you still expose the nitrocellulose membrane at -80C when using phosphoimager instead of a film? I'm also wondering what exposure time your lab uses when using a phosphorimager screen.

    Secondly, I am wondering how many libraries containing different barcodes you can run together in a single flow cells.

    Thank you again Jernej. This protocol has been extremely useful.

    Reply
    Posted by: Anonymous
    November 15, 2011 - 7:49 PM
  18. Cross-linking forms a covalent bond, so is irreversible (read the paper!). -80 would ruin the phosphorimager screen, so don't do it! We normally multiplex ±10 libraries.

    Reply
    Posted by: Anonymous
    November 15, 2011 - 7:53 PM
  19. Cross-linking forms a covalent bond, so is irreversible (read the paper!). -80 would ruin the phosphorimager screen, so don't do it! We normally multiplex ±10 libraries.

    Reply
    Posted by: Anonymous
    November 15, 2011 - 7:53 PM
  20. Cross-linking forms a covalent bond, so is irreversible (read the paper!). -80 would ruin the phosphorimager screen, so don't do it! We normally multiplex ±10 libraries.

    Reply
    Posted by: Anonymous
    November 15, 2011 - 7:53 PM
  21. Hi Jernej,

    In regards to one of the FAQs from Google docs.

    - When analysing PCR products, I see a band corresponding to the size of primer dimers, especially in the sample that was cut low from cDNA gel.

    Yes, it is common to see this band in the sample that was cut low from cDNA gel, and sometimes also in other samples. This is due to contamination from short cDNAs that only contain the sequence of RT primer. If this primer dimer is the dominant product on gel, we advise against sequencing the corresponding sample.

    I seem to be getting this short cDNA contamination all the time. Do you have any advice on how I could try to minimise the contamination? Have you ever isolated fragments of correct-size cDNA from a TBE-urea gel and sent only the isolated fragment for sequencing when you have short cDNA contaminations? Do you think that will work? I think that the concentration of L3 linker that I had used might have been too much. Thank you.

    Reply
    Posted by: Anonymous
    November 22, 2011 - 7:45 PM
  22. There are several possible reasons for this. Maybe one aspect of the protocol is not working, and therefore you are not producing any specific cDNA. If you have no cDNA input, then with enough cycles, you can amplify the primer-primer from any part of the gel. If you are using mammalian cells, try to get the protocol working first with hnRNP C or TIA with Santa cruz antibodies that we used in recent publications. Otherwise, using too much L3 can be a problem.

    Reply
    Posted by: Anonymous
    November 23, 2011 - 4:35 AM
  23. Very useful protocol. I have two questions.

    1. For dephosphorylation of RNA 3'ends, pH 6.5 PNK buffer is used, rather than the pH 7.6 buffer, provided by NEB. Have you compared these two conditions internally?
    ². In the protocol, the final PCR product is not isolated and quantitated before submitting for the sequencing. Are there any potential problems of doing these two steps? Can I isolate the PCR product and re-PCR using the same primers to get more product (for Illumina Hiseq)? Thank you.

    Reply
    Posted by: Anonymous
    January 5, 2012 - 3:40 PM
  24. You can find more related answers in Googledoc http://goo.gl/4tSci, but short answers are also below:

    1. We haven²17;t compared conditions, but increased phophatase activity of PNK at lower pH has been reported in literature, you can read more in the Pubmed ID 1184²1²0.

    ². The PCR product needs to be quantified. We use both qPCR and bioanalyser. Normally, the products of the first PCR should look clean on the gel, otherwise it is a sign of a library that is of low complexity, and is unlikely to generate informative data. Therefore we advise against re-PCR, but it can be done as the last resource.

    Reply
    Posted by: Anonymous
    January 5, 2012 - 4:56 PM
  25. Hi Jernej,

    I noticed you use +/- 10 multiplexed libraries; I was wondering if you knew how many are necessary for a successful run (i.e. to provide sufficient distribution for cluster identification)?

    Reply
    Posted by: Anonymous
    March 29, 2012 - 2:20 PM
  26. The way the primers are designed here, no multiplexing is necessary, because the first three nucleotides in the primer sequence are random (part of randomer = NNN).

    Reply
    Posted by: Anonymous
    March 29, 2012 - 2:28 PM
  27. I appreciate your experiment. I have some qeustions.

    In this protocol, what dŒs barcode do high-throughout squencing?

    I don't understand function of barcode



    Reply
    Posted by: seung kuk P.
    May 23, 2012 - 6:45 AM
  28. Hi,
    this might be a really naive question but I'm wondering at the UV cross linking step, when you say you irradiate once, dŒ's this mean 1 min?

    Thank you!
    Zsofi

    Reply
    Posted by: Zsofia I.
    June 18, 2012 - 1:19 PM
  29. Hello, Thank you for this helpful technique, I just have a question. My experiments protocols are: 1. UV-crosslink RNA-protein; ². Isolate the RNA-protein complex by immunopricitation; 3. Isolate the binding RNA. 3²P-labeling the binding RNA. 4. Analysis the RNA by microarray.
    Because I do not need to sequence the RNA, and I only want to isolate the binding RNA for microarray analysis after UV-crosslink RNA-protein, so I wonder whether I need to do the step 5-7 in your protocols or I could skip from step 4 to step 8 in your protocol?

    Thanks very much, I look forward to your kind reply!

    Sean

    Reply
    Posted by: xiaoyun w.
    July 22, 2012 - 9:09 PM
  30. It is unlikely you will have enough cDNA for microarray hybridisation without some kind of amplification. You can try using steps 4-8, but you could also amplify in other ways.

    Reply
    Posted by: Anonymous
    July 23, 2012 - 6:03 AM
  31. Hi Jernej,

    Is there any published article on how to analyse iCLIP's high-throughput sequencing data? I have just got my sequencing results back following steps in your protocol. I want to make sure I check with you before digging into the data. Thank you.

    Reply
    Posted by: Anonymous
    August 1, 2012 - 10:15 PM
  32. The article is not yet published, but is in preparation by Tomaz Curk ( http://www.fri.uni-lj.si/en/tomaz-curk/), who made a public server: http://icount.biolab.si/. You can contact Tomaz at tomaz.curk@fri.uni-lj.si for more information.

    Reply
    Posted by: Anonymous
    August 2, 2012 - 5:47 AM
  33. Hi,
    it is so powerful technique! But I cannot IP any protein follow protocol. Is there any difference in affinity between different antibodies and their antigen? Could you give me some advice? Maybe we could decrease concentration of SDS or sodium deoxycholate?
    Thanks, I look forward to your kind reply!
    Min

    Reply
    Posted by: Min S.
    August 5, 2012 - 11:04 PM
  34. Hi Min, you can find advice on IP googledoc http://goo.gl/4tSci.

    Reply
    Posted by: Anonymous
    August 6, 2012 - 3:35 AM
  35. Hi Jernej,

    With the barcoding system, I am just wondering if the three random nucleotides are there for indexing purpose during Illumina sequencing run but it's not necessary for splitting the different libraries later on. For RC1, the sequencing results will be something like NNNGGTTNN.... During analysis, do you usually trim the 3-bp from the 5'-end of the results and split the different replicates after the trimming step? I have just realised this was slightly different to the barcoding system used in your NSMB paper. -paul

    Reply
    Posted by: Anonymous
    August 6, 2012 - 9:41 PM
  36. Hi Paul! You can find the answer under the topic of "Use of random barcode in data analysis" in http://goo.gl/4tSci.

    Reply
    Posted by: Anonymous
    August 7, 2012 - 7:58 AM
  37. Hi Jernej,

    I started optimising CLIP couple of months ago and I'm at the stage that I'm convinced that I can efficiently cross link RNA to my protein (checked it by specific qRT PCR). I'm lucky because I don't need to fiddle with the IP since I've optimised before and works fine. But just to double check, after IP and western blotting a smear and a lower amount of original kDa protein is a good sing for cross linking yes?
    So my problems started at the RNase A step, I don't see any changes in size/appearance on WB after treatment... I'm convinced that my protein creates a massive complex (couple of 100 kDa) and it is because my target RNA is 10 kb to start with and there are at least 3 proteins binding to it. I'm working with a RNA virus, that's the explanation for it. I think the reason I don't see any change in kDA is because the complex dŒsn't even enter the gel to start up with. Although I used the given buffer which should break any membrane apart but the proteins are still there possibly protecting the RNA. Did you ever come across similar problems and would you have any suggestions? Also, I understand that the RNase trimming is necessary for the efficient RT step but is it a problem if the RNA is too long? What is too long? DŒs this depend on the RT enzyme used I recon or is this also important for the sequencing?

    I would greatly appreciate yur help because I'm stuck...

    Thank you,
    Zsofi

    Reply
    Posted by: Zsofia I.
    October 10, 2012 - 7:38 AM
  38. Hi Zsofi,

    For partial RNAse digestion we use RNase I (step 3). We use two different concentrations: a lower one that makes fragments with a mean between 50-100 bp and a higher concentration that fragments RNA to around 10bp. The lower one is used for preparing libraries, the higher one is used for analytical reasons.

    The RNAse step is important to (1) allow the protein RNA complex enter the Gel (²) to narrow down the crosslink site to a fragment with a size compatible with high throughput sequencing (maximum around 300 bp). So you definitely need to optimize this step for your experiments.

    If the complex you are studying is not covalently linked it should fall apart during the denaturing Gel run. Only a small fraction of your complex will have all the proteins of your complex crosslinked to the RNA at the same time since crosslinking is a very inefficient step. Therefore with the higher RNAse concentration you should be able to see a radioactive signal at the size of the protein you are studying.

    I hope that helps, best regards,
    Julian

    Reply
    Posted by: Julian K.
    October 11, 2012 - 9:49 AM
  39. Hi Julian,

    I have had some trouble with the RNase step when nuclease-ing the total lysate... In my troubleshooting efforts I read that RNase I is inhibited by 0.1% SDS, which is the concentration used in your lysis buffer. It dŒsn't seem that you guys have any problem though...do you think this is due to using an excess of RNase I or what? Just curiously confused. Thanks,

    sam

    Reply
    Posted by: Sam F.
    February 5, 2013 - 6:00 PM
  40. Hi Sam,

    in our experience the inhibition of RNase I by SDS is not an issue. You just optimize the concentration of RNase I to obtain the desired fragmentation. If you have problems doing that with your buffer conditions, you could also do the RNase digestion on the beads instead of in the lysate.

    Best,
    Julian

    Reply
    Posted by: Julian K.
    February 6, 2013 - 6:19 AM
  41. Thanks for the quick reply Julian. Your recommendation to do the "on bead" digestion is exactly what I have done and it seems to be working fine. Cheers

    Posted by: Sam F.
    February 6, 2013 - 10:12 AM
  42. Hi,

    I was wondering how many minutes have you irradiated the cells in case of HNRNP C?

    Reply
    Posted by: Niaz M.
    November 26, 2012 - 6:29 PM
  43. Hi Niaz,
    we are normally not measuring time of irradiation but the Energy per square centimeter:
    Step 1.²: ... Irradiate once with 150 mJ/cm² at ²54 nm.
    In our Stratalinker this takes 50s. However time of irradiation is not very informative here since it changes with the age or quality of the lamps, etc.
    Cheers, Julian

    Reply
    Posted by: Julian K.
    November 27, 2012 - 6:20 AM
  44. Hi,

    Thank you for wonderful protocol !

    I would like to confirm about adaptor and primer sequences.
    1. L3 adaptor and Rclip RT primer has ²²0;same²²1; sequences, not ²²0;complementary²²1; sequences. Are they O.K.? In my understanding, L3 and TR primers should have ²²0;complementary sequences.
    ². P3 Solexa 3²17; 11 nt sequence (TCTTCCGATCT) looks ²²0;extra²²1;. Both of P5 and P3 have the same sequence, which is complementary to Rclip RT primer or L3 adaptor. I think only P5 should have this sequence.

    Thank you for your help.

    Best,
    Lisa

    Reply
    Posted by: Risa K.
    December 18, 2012 - 3:04 AM
  45. Hi,

    Thank you for wonderful protocol !

    I would like to confirm about adaptor and primer sequences.
    1. L3 adaptor and Rclip RT primer has ²²0;same²²1; sequences, not ²²0;complementary²²1; sequences. Are they O.K.? In my understanding, L3 and TR primers should have ²²0;complementary sequences.
    ². P3 Solexa 3²17; 11 nt sequence (TCTTCCGATCT) looks ²²0;extra²²1;. Both of P5 and P3 have the same sequence, which is complementary to Rclip RT primer or L3 adaptor. I think only P5 should have this sequence.

    Thank you for your help.

    Best,
    Lisa

    Reply
    Posted by: Risa K.
    December 18, 2012 - 3:04 AM
  46. Hi Lisa,

    it is correct that the ends of P3 and P5 primers are the same. This is because of Illumina's primer design for their high throughput sequencing platform. When you look at the 3' end of the Rclip primers (after the Bamhi cleavage site) you can see that they are actually complementary to the 3'end of the L3 adapter.

    Cheers,
    Julian

    Reply
    Posted by: Julian K.
    December 18, 2012 - 10:05 AM
  47. I got it !!!
    Thank you :)

    Best,
    Lisa

    Reply
    Posted by: Risa K.
    December 18, 2012 - 1:11 PM
  48. Hi
    Thanks for the protocol. I have one question that has been bothering me, though. Both the RNA ligase and PNK buffers will expose the antibody column to relatively high dithiothreitol (DTT) concentrations (10 mM and 5 mM respectively). Why dŒsn't this destroy the column by reducing the disulphide bonds holding the heavy and light antibody chains together? Have you ever tried to improve the immunoprecipitation step by attempting to minimize the DTT concentration as much as possible or is this not an issue. Any assistance would be greatly appreciated. Thanks - Greg

    Reply
    Posted by: Greg C.
    February 3, 2013 - 2:13 PM
  49. Hi Greg, we haven't seen an effect of the DTT in the buffers on the IP efficiency, it seems that the concentration is not high enough to reduce the IgG - however, it is worth testing this the first time you do IP, since it is plausible that this will vary dependent on the source of your buffers (company used for PNK and ligase), or antibodies.

    Reply
    Posted by: Anonymous
    February 6, 2013 - 2:45 AM
  50. Hi Jernej,
    Thanks for the reply. The antibody I am using is definitely sensitive to the level of DTT found in the PNK buffer and I need to limit the over-all exposure of the column to DTT as much as possible. As a result, rather than using PNK as the 3' phosphatase, I would like to use an alkaline phosphatase. I noticed that in your ²010 NSMB paper you are using Shrimp Alkaline Phosphatase and in your ²009 Methods paper you use FAST AP. Did you find that the Shrimp phosphatase is significantly better ?

    Thanks again - Greg

    Reply
    Posted by: Greg C.
    February 8, 2013 - 3:52 PM
  51. Hi Greg, we don't have any evidence to suggest that one is better than the other for the on-bead reaction. At the time we were using SAP in the lab generally since it can be heat-inactivated, so therefore we also used it for on-bead (even though here you can't heat-inactivate it on beads). So you can go ahead with either one.

    Reply
    Posted by: Anonymous
    February 9, 2013 - 5:29 AM
  52. I should also add that even though we didn't compare FAST AP and SAP, we did compare SAP with PNK, and we had a lot better results with PNK. It seems that SAP is not efficient as a 3' phosphatase. So it may be better for you to determine the minimal DTT amount in the buffer that is compatible with your antibody, and then continue using it with PNK and ligase. If you use fresh DTT, 1mM is likely to be sufficient both for PNK and RNA ligase.

    Reply
    Posted by: Anonymous
    February 9, 2013 - 5:39 AM
  53. Thanks, I really appreciate the advice.
    -Greg

    Posted by: Greg C.
    February 9, 2013 - 9:03 AM
  54. Hi, thanks for the awesome video. I have two questions related to the reagents:
    1. What concentration is the PEG400? (it only says 4 ul in the protocol).
    ². Under "Reverse transcription", step 6, what is the pH of the TE buffer you use? Is it pH 8?
    Thank you very much for your help. -QT

    Reply
    Posted by: Qiumin T.
    February 7, 2013 - 1:22 PM
  55. Hi QT,
    (1) we are using PEG400 from Sigma (²0²398). It is a viscous liquid.
    (²) Yes, the it is pH 8
    Cheers, Julian

    Reply
    Posted by: Julian K.
    February 7, 2013 - 4:56 PM
  56. Thank you so much Julian. I have another question. Could you recommend a protocol for doing iCLIP with mouse brain tissue? Do you know whether the tissue prep steps from this protocol ( http://ago.rockefeller.edu/Ago_HITS_CLIP_Protocol_June_²009.pdf) will work well for iCLIP as well?

    Reply
    Posted by: Anonymous
    February 13, 2013 - 5:52 PM
  57. This protocol should be fine. We also recently published a bookchapter about the iCLIP protocol which contains information on tissue samples and lots of other useful info and background:
    http://onlinelibrary.wiley.com/doi/10.100²/97835²764458².ch10/summary

    Reply
    Posted by: Julian K.
    February 14, 2013 - 5:19 AM
  58. The pre-publication version of the book chapter is available here: http://www².mrc-lmb.cam.ac.uk/groups/jule/publications/Konig_wiley.pdf.

    Reply
    Posted by: Anonymous
    February 19, 2013 - 1:54 PM
  59. I enjoyed reading about your updated iCLIP protocol in the book Tag-based Next Generation Sequencing from Wiley. I would be grateful for more details about the amount and activity of the 3²-P that you use to radioalabel the RNA. In both the book chapter and the JoVE article I see only volumes, not activities.

    In the figure for step 9, the radiolabel on the 5' end of the RNA is missing, but shouldn't it still be there? At what point in the protocol can we be reasonably sure that we are dealing with unlabeled material?

    Also, have you ever explored non-radioactive approaches to labeling, or is the sensitivity of these methods too low for the purposes of this protocol?

    Thanks!

    John

    Reply
    Posted by: John S.
    June 17, 2013 - 9:23 AM
  60. Dear John,

    with the current protocol most of the radioactivity is gone after the gel purification of the cDNA. You can increase this effect by treating the samples with RNAse after the reverse transcription (The radioactive RNA fragments then running much faster then the cDNAs in the gel). We are currently working on a protocol where we fragment the RNA by alkaline hydrolysis, which will be available soon (We want to avoid using too much RNAse at our desks).

    In addition you should always measure your samples with a Geiger counter. If your final PCRs are still hot, then you should decrease the fraction of beads that go into the labeling reaction.

    Best wishes,
    Julian

    Reply
    Posted by: Julian K.
    June 19, 2013 - 11:52 AM
  61. Hi,
    I have a question which relies on your experience with the data generated with CLIP:
    because of the UV irradiation the protein crosslinks to the RNA which even after proteinase K treatment presents an obstruction to the reverse transcriptase which therefore either skips or adds random nucleotide(s). So my question is that how long the deletions/insertions can be? Is it only one nucleotide or can also be 20?

    Thank you very much!!!

    Reply
    Posted by: Zsofia I.
    August 8, 2013 - 12:06 PM
  62. We see that >80% of cDNAs truncate at the crosslink site, and the mutations are quite rare in the remaining sequences. All we know about crosslink-induced mutations has been published here: http://www.ncbi.nlm.nih.gov/pubmed/22863408.

    Reply
    Posted by: Anonymous
    August 8, 2013 - 12:15 PM
  63. Thanks for your protocol. I have a question about the IgG background signal in the p32 labeled Western Blot. I used mouse IgG1 isotype as a control. I did not crosslink the IgG to the beads, so IgG1 stays around 50 and 25 KDa region. I do observe some radioactivity band around 50 kDa. Do you notice this in your experiments as well? Since it is close to my protein region, can you give me some suggestions to avoid this?

    Sincerely,
    Mei

    Reply
    Posted by: Xuemei Z.
    August 9, 2013 - 1:42 PM
  64. We don't get a signal in control IP. Most likely this is an RBP that non-specifically binds under your conditions. It is important to wash with high-salt buffer, and rotate the tubes for ±5min during these washes. Also, diluting the lysate before IP may help. Standard IP optimisations, basically.

    Reply
    Posted by: Anonymous
    August 9, 2013 - 2:24 PM
  65. Hi,
    Thank you for wonderful protocol.
    Usually how much RNA concentration one should get after Isolation from membrane? I would appreciate your reply.

    Reply
    Posted by: Bhagya B.
    August 13, 2013 - 5:29 AM
  66. Hi,
    Thank you for wonderful protocol.
    Usually how much RNA concentration one should get after Isolation from membrane? I would appreciate your reply.

    Reply
    Posted by: Bhagya B.
    August 13, 2013 - 5:57 AM
  67. Hi,
    I have 2 more questions. In this protocol you did not remove 5' phosphate of the RNA, can you still label the 5' side with P32 by PNK later? Another question, is it possible to just p32 label the RNA, cut the band, extract, degrade the protein and add 3' linker for RT later?

    Reply
    Posted by: Xuemei Z.
    August 21, 2013 - 2:06 PM
  68. Normal PNK has phosphatase activity, so it can replace the 5' phosphate. The original CLIP protocol from Ule et al, Science 2003 added 3' linker after RNA extraction, but as explained in Ule et al, Methods 2005., the efficiency and purity of the protocol increases if linker is ligated on beads.

    Reply
    Posted by: Anonymous
    August 21, 2013 - 2:15 PM
  69. Thanks for this amazing protocol and your rapid and very helpful exchange here in this site.

    Reply
    Posted by: Xuemei Z.
    August 21, 2013 - 2:26 PM
  70. Hello, thank you for this wonderful protocol.
    I have a question:
    -I get positive Radioactive signal at the right size of positive CTRL used in this protocol in the NOT UV samples, it looks exactly as I was using high RNAse condition. why?
    I am phosphorylating the protein? is it possible?
    thank you

    Reply
    Posted by: jessica c.
    February 27, 2016 - 8:45 PM
  71. Hi Jessica, you are right, if you see signal in the non-UV control, this means that the protein is getting phosphorylated in some other way. If it has a kinase domain it may even phosphorylate itself. Or maybe some kinase is getting co-purified? You could check for this by omitting PNK from the phosphorylation reaction.

    Reply
    Posted by: Jernej U.
    March 16, 2016 - 11:07 AM

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